BMP: EXP2 substrate conc and reaction velocity

Question 1

What is the velocity of all reactions dependent on?

Answer 1

substrate concentration

Question 2

What does Michaelis-menten equation relate?

Answer 2

Relates intial velocity of an anzyme catalysed reaction to substrate concentration

Question 3

What is Michaelis-menten equation?

Answer 3

Vi = V_{max [S]} / (k_m + [S])

Vi = inital velocity

Vmax = max velocity of a reaction

Km = the substrate conc at which the enzyme displays half the maximum velocity

S = substrate conc

Question 4

What is the michaelis-menten equation frequently used for?

Answer 4

Determining enzyme-substrate affinity

Question 5

What is the mechanism behind this experiment?

Answer 5

Experiement involves the hydrolysis of the disaccharide sucrose into its monosacchardies glucose and fructose.

Glucose and fructose react with dinitrosalicylate, which on reduction forms a compound which can be measured spectrophotometrically

Reaction catalysed by inveratse

Question 6

Why does sucrose not react with dinitrosalycilate?

Answer 6

It is a non-reducing sugar, where as glucose and fructose are reducing sugars

Question 7

What solution is used for this experiement? What concentration and why?

Answer 7

Equimolar proportions of glucose and fructose (same as hydrolysis products)

0.005M F ; 0.005M G = 10micromole/ ml

Question 8

Describe the procedure

Answer 8

1. Duplicate test tubes prepared with varying volumes of G/F solution, varying volumes of water and 1ml 0.5M sucrose solution (to 3ml)
2. 2ml of dinitrosalicylate added, sealed (parafin), mixed and heated in boiling water for 5mins
3. mixture left to cool and 10ml of water added andmixed
4. measure absorbacne at 540nm
5. plot calibration curve (A/G/F conc)

Question 9

What are the limits of linearity for this experiment?

Answer 9

12 micromol/ml

Question 10

How is the velocity process carried out?

Answer 10

1. Tubes prepared:
   
   • Tube1 (blank): 1ml acetate buffer (7.4) and 1ml Sucrose
   • Tube 2-12: 1ml acetate buffer and 1ml sucrose of diff concs
2. Tubes in hot water bath at 25C for 5 mins to equilibriumize
3. 1ml invertase added
4. after 5 mins at 25C 2ml dintrodalicylate added to terminate enzyme activity
5. 5 mins in boiling water bath and left to cool and 10ml water added
6. Absorbance measured

Question 11

How do you calculate glucose/Frucotse produced after 5 mins?

Answer 11

Using the calibration graph:

Look for absorbance on Y-axis and determine X-value (conc) from that

Question 12

How is glucose/ Frucotse produced per minute calculated?

Answer 12

Dividing previous value by 5

Question 13

What does a michaelis-menten plot look like?

What type of graph is it?

How is Vmax and km calculated?

Answer 13

Question 14

Explain the shapeof the michaelis-menten graph

Answer 14

Velocity of the reaction itially increases with increasing subdtrate concentration. However it levels off as the enzymes become saturated. Eventuall increasing substrate concentration will have no effect on velocity as enzyme concentration is now the limiting factor

Vmax= all enyxmes active sites saturated

Question 15

1. Construct a line-weaver burk graph
2. What type of graph is this?
3. how is Km and Vmax determined?

Answer 15

Question 16

Advantges if lineweaver burk over michaelis menten plot?

Answer 16

More accurate Vmax and Km

Vmax occurs at infintiate substrate and with M plot plato not reached to determine

Question 17

Describe the relationship between substrate and affinity for enzyme

What does a high and low km mean?

Answer 17